Introduction:

chambers (remnants of plant cells) in

slices of cork.

Microbes

Unlike plants and animals are a

heterogenous group of organisms
encompassing animate and inanimate,
prokaryotic and eukaryotic, as well as
unicellular and multicellular biologicl
entities
Can be viruses, bacteria, algae,
protozoans and fungi
Outnumbered the human cells in the body
by 10:1 ratio
Form a community called microbiome
that orchestrate the overall health and
well-being of the human body and may
also be responsible for diseases

Human eye
-

also an optical system having a resolving

power of 0.1mm.

Resolving Power or Limit of Resolution of a

microscope
-

defined as its ability to gather fine details

of the specimen observed
more important than the magnification in
evaluating the usefulness of the
microscope
defined by Abbes (Ernst Abbe)
Equation:

Exercise 1 - The Microscope: Principles,

Parts, Use and Care

Limit of Resolution=

Light Compound Microsope

Numerical Aperture
N . A .
0.61

most familiar laboratory to microbiologists

optical system consisting of aligned lenses
that work together to magnify or enlarge
the image of the specimen under
observation
the first lens that magnifies the image of
the specimen is the objective lens; image
produced is then magnified further by
second lens called the ocular resulting in
much enlarged virtual image
Visible light of the electromagnetic
spectrum ranging from 400nm to 700nm
is used to illuminate this type of
microscope. Thus, its name Light
Compound Microscope.

Microscope
-

can be simple or compound

o Simple Microscope invented and
used by Anton van Leeuwenhoek
and has only one lens.
Anton van Leeuwenhoek was the first to
see bacteria, yeasts, protists, sperm and
blood cells in 1673 using a 300x simple
microscope.
o Compound Microscope invented by
Zacharias Janssen in 1595 and has
more than one lens.
Robert Hooke was used a compound
microscope and was the first one to
introduce the word cell in scientific
literature 1665 describing the numerous

0.61
sin

0.61 is Abbes constant derived from light

coherence and visibility
(lambda) is the wavelength of light
illuminating the microscope can be altered
using special filters. Wavelength of
visible white light is 550nm (indirect
sunlight). Other electric light sources are
tungsten (light bulb), halogen lamp and
fluorescent lamp.
200nm range ultraviolet light
Higher than red infrared light
(eta) is the refractive index of the
medium between objective lens and glass
slide.

Values of :
Air
Water
Linseed Oil
Cedar Wood Oil
Canada Balsam

1.00
1.33
1.48
1.50
1.52

(theta) is the half the value of the

angular aperture. The Angular
Aperture is also known as the cone
angle; it is the cone angle that enters
the objective lens.

The closer the condenser to the specimen;

the bigger is the cone angle.

Objective
45x dry
high power
objective
100x oil
immersion
objective

Cone
Angle or
Angular
Aperture

(theta)

64

32

116

58

Another datum that is usually engraved

adjacent to the Tube Length is the
recommended thickness of the
coverslip to minimize spherical and
chromatic aberrations.
Coverslips have thickness ranging from
0.11mm to 0.23mm. 0.17mm is the
standard recommended thickness of the
coverslip in mm.
o Working Distance distance between
the objective lens and the
coverslip/slide when specimen is in
focus. The longest is under the LPO and
the shortest is under the OIO.
For the OIO, 1.00 is the objective
magnification, 1.25 is the numerical
aperture, 160 is the tube length and 0.17
is the recommended thickness of the
coverslip.
o Tube Length distance from the
nosepiece-objective junction to the
observation tube where the ocular is
inserted. It has a standard value of
160mm for most microscopes as set by
the Royal Microscopical Society of
London.
o Xylene solvent of choice to clean
hardened oil sticking from the Oil
Immersion Objective.

Microscopi
c
Parameter
s
N.A.
Half
Angular
Aperture
()
Angular
Aperture
(2x)

LPO

HPO

Resolving
power in
um

1.34um

0.52um

0.27um

Light intensity
Build-in light bulbs for microscopes can be:

Tungsten (Incandescent)
Incandescent like the common household
light bulb is used for less expensive
microscopes; it warms up easily and
produces less intense yellowish light.
Fluorescent
Fluorescent produces more intense white
and cooler light.
Halogen
Halogen provides brilliant light, uniform
illumination and longer life-span.

Arc lamps:
-

mercury vapor
xenon
zirconium

Parts of the Light Compound Microscope

OIO

0.25
14.48O

0.65
40.54O

1.25
56.44O

28.96O

81.08O

112.89O

a. Eye piece or ocular lens where you

look through in a microscope
b. draw tube tube where the eyepiece is
attached
c. dust shield the mechanical part where
the revolving nosepiece is attached;
protects the objectives from dust
d. revolving nosepiece mechanical part
where the objectives are attached; can be
rotated to position the objective in use.
Also called Turret

e. Objectives the lenses above the stage

attached to the revolving nosepiece are
Scanner 4X with numerical
aperture of 0.1
Low Power Objective (LPO)
10x with numerical aperture of
0.25
High Power Objective (HPO)
marked 40x and sometimes called
the Dry High Power
Oil Immersion Objective
marked 100x
f. Stage platform under the objectives
where you place the specimen to be
viewed.
g. stage aperture the opening at the
stage where light passes through from the
condenser to the objectives.
h. condenser the lens located below the
stage collects light and delivers it to the
objectives in the form of a cone of light
whose angle is known as the angular
aperture
i. iris diaphragm the mechanical part
below the condenser which can be closed
or opened with its lever to regulate the
amount of light that gets to the condenser.
Some old microscopes have the annular
type, different sizes of hole under the
microscope.
j. mirror the part that reflects light from
the outside to illuminate the field of vision
k. mirror fork the adjustable part that
holds the mirror
l. base the rectangular of U-shaped part
that supports the body of the microscope
m. arm the C-shaped part where the
microscope is grasped when it is moved or
transported
n. fine adjustment - the knob at the body
tube used to focus the specimen viewed
under the LPO
o. coarse adjustment the knob at the
body tube used to focus the specimen
viewed under the LPO
p. condenser adjustment the knob at the
pillar of the microscope used to adjust the
contrasting level of the condenser
q. pillar the part connecting the body of
the microscope to the base
r. inclination joint the joint at the pillar
used to tilt the microscope for convenient
observation

The microbial world largest assembly of a

wide variety of organisms occurring nearly
everywhere in nature. Major group of organisms
include:
Prokaryotes
Eukaryotes

Bacteria
Cyanobacteria (bluegreen algae)
Yeasts, molds,
protozoa, algae
Viruses

Viruses
-

non-living DNA or RNA nucleoprotein

complex exhibiting an obligatory
intracellular parasitic nature

In the classification of living organisms,

there are currently 6 proposed
kingdoms and 3 domains.

6 kingdoms

Archaea

Bacteria

Protista

Animalia

Plantae

Fungi

Prokaryotes that do
not have
peptidoglycan in
their cell wall
Prokaryotes which
have peptidoglycan
in their cell wall
Unicellular eukaryotes
comprising the
protozoans, algae
and slime molds
Multicellular
eukaryotes which
takes in their food by
ingestion
Multicellular
eukaryotes which are
photosynthetic
Multicellular nonchlorophyllous
eukaryotes which
performs
extracellular
digestion and then
absorbs their food
from the outside

3 Domains
Bacteria, Archaea, Eukarya ( BAE )

Exercise 2: Micrometry and the Microbial

World

The most recent form of microbiological hazard to

be recognized are the small proteinaceous
infectious particles are called PRIONS lack

nucleic acid and VIROIDS small circularly

closed RNA molecules infecting plants. Except for
these and the viruses and some diseaseproducing species of microorganisms, more
species perform important and beneficial roles in
the grand scheme of biosphere. The prokaryotes
and the eukaryotes are, in general, viewed under
the compound microscope either in their living or
fixed/stained state. This process facilitates
observation of their morphological and in some,
locomotory properties.
Lantern slides
-

rectangular glass the size of your palm

containing photographic images of
bacteria contained in the microscopic field
Cell Shape

Groupings

Cell Structure

Bacilli
Cocci
Spirals
Singly
In Pairs
In Chains
Tetrads
Clusters
Capsule
Flagella
Endospore
Metachromatic
granule

Exercise 3: Preparation and Sterilization of

Culture Media and Glasswares
The study of microorganisms in the laboratory
require them to be grown or cultured in an
axenic (pure; presence of a single species)
state. This condition is achieved by cultivating
them in artificial chemical preparations
referred to as culture media.
Culture media
-

usually come in powder form containing

essential ingredients for the metabolism of
microorganisms (macronutirents,
micronutrients and trace elements)
pH indicators for the cultivation of
microorganisms
can be prepared in liquid form called
Broth or in solid form called Agar
The presence of agar is therefore a
solidifying agent. Without it the culture
medium remains in liquid form.
need to be sterilized by autoclaving,
heating or membrane filtration when it is
prepared.
If it is not sterilized, then ubiquitous
unwanted microorganisms called
Contaminants may proliferate and grow
in them thereby spoiling them beyond
use.

Sterilization
Calibration of the ocular micrometer with a
stage micrometer

the process of killing all forms of life

including heat-resistant bacterial
endospores and viruses
it is achieved mainly through moist and
dry heat

The former uses steam under pressure to achieve

temperatures exceeding those of boiling water.
This in turn kills contaminants.

Calibration Factor

SM x 1000 m
=m per ocular unit
OM

Other units can also be used

1000 m=10 microns=0.01 mm

most common method of moist

Autoclavin
heat sterilization
g
uses to sterilize both glasswares
and culture media
uses the principle of convection:
heat transfer via air
Dry heat process takes longer since air is
sterilizatio
not an effective conductor of
n
heat compared to steam
only recommended for sterilizing
glasswares
Membrane uses special filters with known
Filtration
pore size that will not allow
the passage of contaminants
used only for heat sensitive

media components such as

amino acids and sugars
Conditions during sterilization
(autoclaving):
1. 15 psi or 118 KPa
2. 121C(250F) steam that corresponds to
internal temperature
3. 15 to 20 minutes in the autoclave
Preparation of Culture Media

Culture media comes from dehydrated

form first > in order to rehydrate distilled
H2O (deionized) is used
Distillation = to remove other inorganic
chemical compounds o remove unwanted
ions

1. Nutrient Agar desired amount is 200mL

2. Nutrient Broth 100mL
3. Potato Dextrose Agar 100mL
4. Sabourauds Dextrose Agar 100mL
Computation

As long as asepsis is observed in every

transfer, no problems should arise in the
maintenance and handling of pure
cultures.

Aseptic technique is a must for all activities

that involve culturing and isolating desired
microorganisms. It starts from:
a. sterilization of glasswares
b. preparation of media
c. culturing and subculturing
Media preparation Sterilization and
Inoculation
Sterilization
-

voiding all microorganisms

glasswares are sterilized at 160O to 180O in
hot air convection oven for 2-3 hours

Disinfection
-

can apply to inanimate objects

kills pathogenic microorganisms

Antiseptic
-

living tissues

For example, N.A. container states that 39g:1L

then..

Sanitization

V1/V2 = W1/W2 -> 1000mL/100mL = 39g/x

Culture media

X = V2 (W1) over V1

X = 3.9g
Important Reminders
1. If large amount of medium is to be weighed,
use a Triple Beam Balance
2. Make sure that media bottles are closed when
not in use as many are hygroscopic and will
absorb humidity and solidify if kept exposed to air
3. For broth media, heating is not necessary
simply mix or stir with water
Exercise 4: Aseptic Technique/Transfer of
Cultures
Pure cultures (axenic) are an indispensable
aid in the study of microorganisms because
in microbiology one deals with a population of
cells and all of them must be mirror image (clone)
of one another. Hence, all attempts must be
made to prevent the inclusion of unwanted
microorganisms (contaminants) which can
easily come from the immediate environment like
from the air.

reduce germs/microorganisms

a nutrient material prepared for the

growth of microbes in a laboratory

Inoculum
-

microbes that are introduced into a

culture medium to initiate growth

Culture
-

microbes that grow and multiple on a

culture medium
comprises of:
1. Red Algae (Gracilaria and Gelidium)
2. Agar - made out of algae/ Gelidium
Ghalaria - solidifying agent
3. Peptone - Product of enzyme digestion
proteins - Provides peptide for bacteria
4. Trace elements (K, P, Fe, S)
5. Yeast Extract - contains vitamins,
coenzymes and nutrients
6. Casein Hydrolase - used to enrich
media
7. pH indicators - for basis to know if
media(&reaction) is due to an acid or
base

8. serum, whole blood(?), heated whole

blood(?)
Heated Whole blood - Sheeps
blood
Types of media:
1. Synthetic medium/Chemically defined
medium
- medium prepared in the laboratory for
materials of precise or reasonably welldefined composition
2. Complex medium
- contains reasonably familiar materials by
vary
3. Selective medium
- encourages the growth of some organisms
but suppresses the growth of other
4. Differential medium
- has a constituent that causes in a color
change or change in pH
5. Enrichment culture
- contains special nutrients that allow the
growth of a particular organism that might
not otherwise

Pure Culture Technique which means

that it is used to purify and ensure of pure
(axenic cultures) from mixed cultures
o Interrupted flaming of the loop
before doing a streaking process
o Multiple streaking is done in four
quadrants on the agar plate

Media used
Gram-positive (+) bacteria
1. Staphylococcus aureus

Broth 5mL
Slant 6mL
Butt-slant 6mL
Deep/Stab/Butt 5mL

Agar plate 15-20mL

2. Bacillus subtilis
3. Yeast (Saccharomyces cerevisiae)
Gram-negative (-) bacteria
1. Escherichia coli
Facultative Anaerobic can withstand air in the
medium specifically in broth

Setting the tube

a. For stabs or butts: let it solidify upright
b. For slants: set tubes in a slanted
position
c. For butt slants set tubes in a semislanted position
Broth medium
Agar Plate
Stab/deep/butt agar
Butt/Slant

Inoculating loop
Inoculating needle

Inoculating loop or
needle
Never put the cotton plug in table as
this will cause contamination
Slant

Interrupted Multiple Streaking (Multiple

Interrupted Overlap Technique)

B. subtilis endospores are refractor cells that can

transform from vegetative cell -> dormant cell
and withstand extreme temperature
Colony pure culture that originate from a single
cell; visible mass of billions of cells

Exercise 6 Isolation of Microoragnisms

From Environmental Samples
Microbial life forms are distributed
everywhere
Found widespread in the ecosphere the
atmosphere (gas), hydrosphere (water),
and lithosphere (rocks and soil)
1) Basic nutrient supply
Most bacteria can live under some basic
conditions as follow.
Exercise 5 Quantitative Analysis of
Microbial Populations Through Standard
Viable Plate Count Methods
Microorganisms studied in
Nature - In vivo
Laboratory - In viro
Methods in Microorganism Isolation
Pour Plate Method - Pour plating is usually used
when you want to determine CFUs/ml (quantify a
bacterial stock for example) or PFUs/ml (quantify
a phage stock). Once it's pour plated, you can't
really dig the bacteria out of the agar.
Spread Plate Method - Spread plating is used
when you want to isolate specific clonal colonies,
for example, after cloning to select for cells that
uptook a plasmid.
VALID COUNT FOR COLONIES *30 - 300 colonies
HIGH DILUTION = LOW NUMBER OF COLONIES
FORMED
Decontamination to kill the bacteria in the petri
dish
CFU/mL = Average number of colonies x inverse
dilution level
________________________________________________________________

Volume of cell suspension

plated

(A) Photosynthetic bacteria require light,

water and necessary mineral only.
(B) Chemosynthetic bacteria require water
and mineral and the specific substrates to
maintain their life.
(C) Parasitic and saprophytic bacteria live
outside living cells. Therefore, if we can
supply some basic conditions, they can
multiply rapidly. Parasitic bacteria live inside
the bodies of the hosts, but outside the cells,
so, we need to know the detail requirement,
then, the bacteria can be cultivated. When we
know their living conditions, then, we can
control these bacteria and keep our bodies or
cattles healthy.
(2) Suitable temperature The sizes of
bacteria are very small, so, they cannot
control the temperature in environment.
Also, their metabolism would be affected
by the environmental temperature. If the
environmental temperature matches the
optimal temperature of their enzymes
activities, the metabolic rate of the
bacteria can be largely raised. Then, the
bacteria can multiply rapidly. According to
the temperature requirement, bacteria can
be divided into the following three
categories.
(A) Bacteria that live in low temperature,
they live in the temperature between 0oC to
30oC. But usually, the temperature between
10oC to 15oC would be the most favourable.
This bacteria live in deep sea or in
refrigerators.

(B) Bacteria that live in medium temperature,

they live in the temperature between 15 oC to
55oC, and normally, take 25oC to 37oC as the
most favourable. This bacteria are parasitic in
warm-blooded animals.
(C) Bacteria that live in high temperature,
they live in the temperature between 25 oC to
90oC. The most favourable range is from 50 oC
to 60oC. This bacteria live in hot springs or
near the hot brine vent in deep sea.
(3) The suitable acidity, Most bacteria can
live in the enviornment with pH range
from 6.5 to 7.5. The exect pH would be
dependent on the actual environment they
live. For example, the bacteria living in
blood would like the pH at 7.3, because
the pH of blood is 7.3. The bacteria in
alimentary canal would like an alkaline pH,
because the pH inside the alimentary is
alkaline. The bacteria in the stomach
would like to have an acidic pH, because
the environment in the stomach is acidic.
(4) Proper humidity, Bacteria require a
certain humidity to keep their lifes. As
some bacteria need to live in water. But
most
bacteria
prefer
a
humid
environment. If the environment is humid,
it would be easier for bacteria to keep the
vegetative bodies.
If the environment is very dry, most
bacteria cannot exist in the active
vegetative forms. Some bacteria would
become condensed and changed into the
endospores.
Endospores
can
resist
drought for a long time in order to survive
over the unfavourable environment. When
the weather becomes moist again, they
absorb water and restore the vitality. In
such cases, it would be very difficult to kill
all bacteria. Such bacteria endospores can
exist in the form of powder and can
become airborne. If such situation can
occur, the type of bacteria have necessary
condition to become the biological
weapon, as the Bacillus anthracis.
(5) Proper water potential, A proper water
potential is necessary for the growth of
bacteria. If the medium is isotonic to the
bacteria cytoplasm, the bacteria would
grow rapidly.

If the water potential of the medium is low,

water would flow out of the bacteria and
the metabolism of the bacteria would be
suspended. Therefore, the addition of
concentration solution can be a way of
food preservation. As the use of brine and
honey etc. (6) Proper oxygen supply,
Some bacteria require free oxygen, as the
cultivation of Corynebacterium diphtheriae
and Vibrio cholerae require free oxygen,
but the cultivation of Clostricium tetani
and Treponema pallidum should not have
free oxygen.
(7) Proper illumination, some
photosynthetic bacteria require sunlight
illumination for metabolism. But the ultraviolet radiation in sunlight can inhibit the
replication of DNA. So, sunshine can be
used as a means of bacteriostasis.
Bacteriostasis is the process to inhibit the
multiplication of bacteria, but bacteria is
not killed. If the fixed bacteria receive
sunlight of other wavelength, it can
restore the vitality. It is called the
photoactivation. Therefore, chinese people
would like to put clothes in sunshine
before storage.
(8) Other special needed, some bacteria
would have special needs for their growth.
If the bacteria is normally living in blood,
then, we need to add some blood serum
to cultivate it.
(Retrived from http://www.lungtp.com/
prokaryota/e_decce.html on March 12, 2016)
A. Airborn Microorganisms
1. N.A. plates are suitable for isolation of bacteria
and SDA for molds and yesrs
2. Expose the plates to the suorrounding air by
opening NA plates for 1-2 minutes
3. Incubate the plates at 35OC for 24 hours
B. Microoragnisms in the Soil and Pond
Water
1. For soil sample, weigh 1g soil and dilute in
99mL sterile distilled water
2. Transfer 0.1mL of soil suspension in petridishes
containing strength NA medium

C. Microorganiss in Human Skin

4. Freezing and storage in liquid nitrogem

1. Swabbing is used

Drying of Cultures In Soil effective

method of preservation for bacteria
belonging to the genera Bacillus subtilis,
Clostridium tetani and Streptomyces and
other spore-forming bacteria
Storage in Sterile Distilled Water
very simple and inexpensive method
which is recommended for fungi, yeasts,
plant pathogenic, actinomycetes etc with
an average storage life of 5 years

2. Incubate culture plates at 37OC for 24 hours

Exercise 7 Purification, Maintenance and
Preservation of Microbial Cultures
Pure culture technique involves use of
any procedure in which a given species of
organisms mixed with other organisms in
a particular environment, is separated and
cultivated as a pure culture
Pure Culture composed of population of
cells derived from a single cell
Streak Plate Method useful isolation
so that even if the initial steaks yields
confluent growth, well-isolated colonies
develop along the lines of later streaks
after incubation

Clonal Population nutrient-based culture

medium

Four common methods used to maintain

pure cultures
1. Periodic transfer to a fresh medium
2. Overlaying slant cultures with mineral oil
3. Freeze-drying (lyophilization)

Composed of millions of bacterial

cells of a specific bacterial species
derived from a single cell
These bacteria are inculated on
liquid culture medium, referred to as
a broth culture, or on solid medium,
either as slant culture or as a plated
culture
Incubate slants and agar plates at
35OC for 24 to 48 hours